Bioconjugates such as oligonucleotide-enzyme conjugates are employed in a wide variety of molecular biology applications and diagnostic assays. Such conjugates have traditionally been prepared by a variety of methods, such as glutaraldehyde crosslinking, maleimide-thiol coupling, isothiocyanate-amine coupling, and Schiff base formation/reduction. Each of these procedures involves multiple steps that require the enzyme, oligonucleotide, or both, to be modified with the appropriate linking moiety and then purified before being combined and reacted with each other. Often the modification reaction results in an unstable reactive enzyme or oligomer intermediate that must be purified and used immediately. For these and other reasons the yield of conjugate is highly variable when these techniques are used. Furthermore, a large excess of oligonucleotide is usually required, reaction times are lengthy, and several purification steps are needed to obtain a purified conjugate. Finally, in most instances a portion of the enzymatic activity is lost due to the nature of the chemical reactions, lengthy reaction times, and numerous purification steps.
One method of making peptide-protein conjugates utilizes carbodiimide activation of carboxyl residues on the protein to facilitate coupling with primary alkyl amino groups of the peptide (see Hermanson, Bioconjugate Techniques, Academic Press, 1996). Coupling occurs at a pH range of 4.7-7.0. The author notes that nonspecific side reactions such as self-polymerization of the peptide and protein are common under the conditions necessary to produce appreciable amounts of the desired conjugate. Moreover, it is often necessary to drive the reaction by employing a large relative molar excess of either the peptide or protein to be coupled. The need to use a large excess of peptide is likely due to protonation of the primary amino groups under the pH conditions (&lt;7) required to activate the carboxylic acid groups by the carbodiimide. Thus, under low pH conditions only a small fraction of the peptide molecules present possess amino groups which are unprotonated and reactive towards the carbodiimide-activated carboxyl moieties of the protein. Furthermore, peptides which possess more than one amino group may become crosslinked to each other and to the protein at multiple sites. Crosslinking often alters the structure of a peptide so that its ability to serve as an immunogen or ligand in a diagnostic assay is compromised.
Synthetic oligonucleotides which contain a primary amino group are useful for preparing hybridization probes and may be linked to enzymes by a variety of methods as described by Hermanson (ibid). However, no discussion is made of using carbodiimides to activate protein carboxyl groups for direct in situ reaction with amino derivatized oligonucleotides. It is believed that primary amino groups on synthetic oligonucleotides are protonated and unreactive under the low pH conditions necessary to activate protein carboxyl groups. Thus, efficient carbodiimide mediated conjugation of an amino derivatized oligonucleotide to a protein is not possible.
For these reasons direct conjugates are expensive and difficult to make with reproducible results. This has prevented them from becoming commonplace tools in molecular biology and diagnostic applications despite the promise they hold for improving assay sensitivity and simplifying nucleic acid detection schemes.